A cDNA clone was isolated from a mouse pre-B cell line, the sequence of which has a very high homology with rat and human thymosin beta 4 genes. However, the mouse clone has an insertion of 98 bp relative to the published rat and human sequences upstream of the coding region. By isolation of a second set of clones from a different cDNA library and by cloning a PCR amplified region of mouse genomic DNA it was confirmed that the insertion is not a cloning artifact. Furthermore, it was shown by RNase protection assays with RNA from the pre-B cell line that two sizes of thymosin beta 4 mRNA exist, a long form containing the 98 nucleotide insertion, and a short form that corresponds to the known rat and human mRNA. The short form is about 50 times more abundant than the long form. Analysis of genomic DNA by sequencing and Southern blotting revealed that both forms are encoded by a single gene in the mouse. The two forms of mRNA arise by differential RNA splicing; the long mRNA contains three separate exons, whereas the short mRNA is missing exon 2. The long mRNA is present in two different pre-B cell lines, spleen and thymus, but could not be detected in brain, liver, and kidney. It is possible that the longer mRNA, which encodes a hydrophobic NH2-extension of six additional amino acids, plays a role in lymphocyte function or development. In contrast to the mouse which has a single thymosin beta 4 gene, rat and human have multiple homologs. Most or all of these also contain sequences that cross-hybridize with the newly discovered exon 2. A polymorphic thymosin beta 4 gene has been found in human DNA.